Chlorine dioxide gas mediated inactivation of the biofilm cells of.

This study evaluated the chlorine dioxide (ClO2) gas mediated inactivation of the biofilm cells of foodborne pathogens on food contact surfaces. Biofilm cells of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes were developed on stainless steel (SS) and high density polyethylene (HDPE) coupon surfaces, and 5-day-old biofilms were treated with ClO2 gas at 60 and 90% relative humidity (RH) for up to 20 min. With an increase in gas concentration and treatment time, significant differences (p < 0.05) were observed between reduction levels under different RH conditions. Treatment with 50 ppmv of ClO2 gas (60% RH) for 20 min resulted in log reductions from 2.08 to 4.62 and 2.08 to 4.41 of the biofilm cells of three pathogens on SS and HDPE surfaces, respectively. The levels of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS and HDPE surfaces were reduced to below the detection limit (0.48 log CFU/cm2) within 15, 20, and 20 min, respectively, when exposure to 50 ppmv of ClO2 gas at 90% RH.

Antimicrobial mechanism of luteolin against Staphylococcus aureus and Listeria monocytogenes and its antibiofilm properties.

Luteolin (LUT) is a naturally occurring compound found in a various of plants. Few recent studies have reported LUT antimicrobial activities against bacterial pathogens, however, the fundamental LUT mediated antimicrobial mechanism has never been elucidated. This study aimed to investigate the antimicrobial activities of LUT and its mode of action against Staphylococcus aureus and Listeria monocytogenes, either as planktonic cells or as biofilms. Here, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of LUT against S. aureus and L. monocytogenes were determined using the broth microdilution method, and the antimicrobial mode of LUT was elucidated by evaluating the variations in both cell membrane integrity and cell morphology. Moreover, the biofilm inhibition was measured by crystal violet staining assay, while its qualitative imaging was achieved by confocal laser scanning microscope and field emission scanning electron microscope. MIC and MBC values of LUT against S. aureus were 16-32 and 32-64 µg/mL, and 32-64 and 64-128 µg/mL for L. monocytogenes. LUT destroyed the cell membrane integrity, as evidenced by a significant increase in the number of non-viable cells, and well-defined variations in cell morphology. Moreover, LUT presented robust inhibitory effects on the biofilm formation, enhanced antibiotics diffusion within biofilms and killed efficiently mono- and dual-species biofilm cells. Overall, LUT demonstrates potent antimicrobial properties on planktonic and biofilm cells, and the biofilm formation, and thus has the potential use as a natural food preservative in foods.

Actively detached Pseudomonas aeruginosa biofilm cell susceptibility to benzalkonium chloride and associated resistance mechanism.

The present work aimed at studying physiological properties of Pseudomonas aeruginosa cells actively detached from biofilm formed on stainless steel and comparing them with their planktonic counterparts as a function of growth temperature (20 °C and 37 °C). The susceptibility of P. aeruginosa cells to benzalkonium chloride (BAC) was studied. Furthermore, the effect of BAC on the cell membrane integrity and the role of the cell membrane fluidity in the cell-scale-resistance mechanism were investigated. Our results showed that actively detached biofilm cells were more susceptible to BAC treatment than planktonic ones. A greater leakage of intracellular potassium after BAC addition was observed in actively detached biofilm cells, which reflects their membrane vulnerability. The rise of the growth temperature from 20 to 37 °C increased the membrane rigidity of planktonic cells comparatively to their actively detached biofilm ones. Under experimental conditions developed in this work, our data highlighted that actively biofilm-detached and planktonic P. aeruginosa cells have distinguishable phenotypes.

Explorative gene analysis of antibiotic tolerance-related genes in adherent and biofilm cells of Pseudomonas aeruginosa.

BACKGROUND: Antibiotic tolerance has attracted worldwide attention, as it leads to chronic, refractory, and persistent infections that are difficult to control. Bacterial biofilms are well known to be more tolerant to antibiotics compared to planktonic bacteria. We previously revealed that adherent bacteria on a solid surface also exhibited tolerance to antibiotics before forming a biofilm. However, little is known about the mechanisms of antibiotic tolerance for adherent or biofilm cells. OBJECTIVES: We investigated the mechanisms of antibiotic tolerance in the biofilm life cycle using adherent and biofilm cells, and evaluated the possibility that common mechanisms operate at each stage. METHODS: We constructed transposon mutants of Pseudomonas aeruginosa PAO1 and screened for low-tolerant mutants with two different methods, using adherent cells and biofilm cells. RESULTS: Fourteen and nine mutants exhibiting low antibiotic tolerance were detected in the adherent cells and biofilm cells, and 14 and 7 candidate genes linked to this tolerance were identified by sequencing, respectively. Eight of the 14 genes related to the antibiotic tolerance of the adherent cells were involved in biofilm formation. Two of the seven genes related to the antibiotic tolerance of biofilm cells participated in the antibiotic tolerance of adherent cells. CONCLUSIONS: The antibiotic tolerance of adherent cells and biofilm formation appear to be under the same regulation mechanism to promote survival in the presence of antibiotics. Antibiotic tolerance shows a complex regulation mechanism at each stage of biofilm formation.

Candida glabrata's recurrent infections: biofilm formation during Amphotericin B treatment.

UNLABELLED: Candida species are responsible for recurrent human infections, mostly in immunocompromised patients, due to their high vulnerability. Candida glabrata has a major role in systemic candidiasis and Amphotericin B (AmB), a polyene only used in hospitals, is frequently used to treat this disease. Lately, however, clinical evidences of Candida recurrent infections during these treatments are being described, probably due to biofilm (re)formation during this therapy. Thus, this work aims at inferring if C. glabrata biofilms are still being formed during AmB treatment. For that, C. glabrata biofilms were formed in the presence of AmB and analysed by dry weight. Matrix composition was analysed quantifying carbohydrates and, specifically, ß-1,3 glucans. Results demonstrated that, although in a lesser extent, C. glabrata is able to develop biofilms in the presence of AmB, with a thick extracellular matrix, with an increase on carbohydrates, especially ß-1,3 glucans. Therefore, it is confirmed that complex biofilms of C. glabrata can be formed during an AmB treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows new insights regarding recurrent candidiasis. The authors demonstrated that Amphotericin B did not totally prevent the development of biofilms during Candida glabrata's infection treatment and that the change in the biofilm matrices may have a high responsibility for the fail in the treatment of systemic candidiasis.

Resistance, Tolerance, Virulence and Bacterial Pathogen Fitness-Current State and Envisioned Solutions for the Near Future.

The current antibiotic crisis and the global phenomena of bacterial resistance, inherited and non-inherited, and tolerance-associated with biofilm formation-are prompting dire predictions of a post-antibiotic era in the near future. These predictions refer to increases in morbidity and mortality rates as a consequence of infections with multidrug-resistant or pandrug-resistant microbial strains. In this context, we aimed to highlight the current status of the antibiotic resistance phenomenon and the significance of bacterial virulence properties/fitness for human health and to review the main strategies alternative or complementary to antibiotic therapy, some of them being already clinically applied or in clinical trials, others only foreseen and in the research phase.

Clinical and microbiological features of host-bacterial interplay in chronic venous ulcers versus other types of chronic skin ulcers.

Introduction: Chronic venous ulcers of the lower limbs develop in the context of advanced venous disease and have a significant impact on the patient's quality of life, being associated with depression and worrisome suicide rates, as well as with an economic burden caused by increased medical care costs and high epidemiological risks of healthcare associated infections and emergence of strains resistant to multiple classes of antibiotics and/ or antiseptics. Although numerous studies have investigated the composition of the chronic wounds microbiome, either by culture-dependent or independent methods, there are no data on the association between virulence and resistance profiles of strains isolated from venous ulcers and the clinical picture of this pathology. The elucidation of pathogenic mechanisms, at both phenotypic and molecular level, is crucial in the fight against these important human microbial agents, in order to develop novel biomarkers and discover new therapeutic targets. Methods: In this study we aimed to characterize the phenotypic virulence profiles (including the ability to develop biofilms) of microorganisms isolated from chronic skin wounds and to correlate them with the clinical symptomatology. Considering the high incidence of Staphylococcus aureus infections in chronic ulcers, but also the ability of this species to develop multi-drug resistance, we performed an more in-depth study of the phenotypic and genotypic virulence profiles of methicillin-resistant Staphylococcus. Results: The study revealed important differences regarding the clinical evolution and virulence profiles of microorganisms isolated from lower limb wounds, as well as between patients diagnosed with chronic venous ulcers and those with lesions of different etiology.

Activity of disinfectants against foodborne pathogens in suspension and adhered to stainless steel surfaces.

The purpose of this study was to investigate and compare the efficacy of various disinfectants on planktonic cells and biofilm cells of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Numbers of viable biofilm cells decreased after treatment with all tested disinfectants (iodine, biguanide, quaternary ammonium compounds, peracetic acid and sodium hypochlorite). Sodium hypochlorite was the most effective disinfectant against biofilm cells, while biguanide was the least effective. Scanning electron microscopy observations revealed that cells adhered on stainless steel surface after treatment with the disinfectants. No viable planktonic cells were observed after treatment with the same disinfectants. Based on our findings, we concluded that biofilm cells might be more resistant to disinfectants than plancktonic cells.

Physiological changes and growth behavior of Corynebacterium glutamicum cells in biofilm.

Biofilm cells are well-known for their increased survival and metabolic capabilities and have been increasingly implemented in industrial and biotechnological processes. Corynebacterium glutamicum is one of the most widely used microorganisms in the fermentation industry. However, C. glutamicum biofilm has been rarely reported and little is known about its cellular basis. Here, the physiological changes and characteristics of C. glutamicum biofilm cells during long-term fermentation were studied for the first time. Results showed that the biofilm cells maintained stable metabolic activity and cell size was enlarged after repeated-batch of fermentation. Cell division was slowed, and chromosome content and cell proliferation efficiency were reduced during long-term fermentation. Compared to free cells, more biofilm cells were stained by the apoptosis indicator dyes Annexin V-FITC and propidium iodide (PI). Overall, these results suggested slow-growing, long-lived cells of C. glutamicum biofilm during fermentation, which could have important industrial implications. This study presents first insights into the physiological changes and growth behavior of C. glutamicum biofilm cell population, which would be valuable for understanding and developing biofilm-based processes.

Comparative Study on the Impact of Growth Conditions on the Physiology and the Virulence of Pseudomonas aeruginosa Biofilm and Planktonic Cells.

The aim of the present work was to study and compare the effect of growth temperature (20, 30, and 37°C) and surface type (stainless steel and polycarbonate) on the production of virulence factors, such as proteases and siderophores, and the risk of surface contamination associated with Pseudomonas aeruginosa biofilm and planktonic cells. The increase of growth temperature from 20 to 37°C increased (approximately twofold) the electronegative charge and the hydrophobicity of the P. aeruginosa biofilm cell surface. P. aeruginosa biofilm cell adhesion to stainless steel and polycarbonate was 5- and 1.5-fold higher than their planktonic counterparts at 20 and 30°C, respectively. The increase of growth temperature from 20 to 37°C increased the production of proteases (twofold) and siderophores (twofold) and the cytotoxicity (up to 30-fold) against the HeLa cell line in the supernatants of P. aeruginosa planktonic and biofilm cultures. This study also highlighted that biofilm and planktonic P. aeruginosa cells exhibited distinct physiological properties with respect to the production of virulence factors and the cytotoxicity against the Hela cell line. Therefore, effective disinfection procedures should be adapted to inactivate bacteria detached from biofilms.

Antibacterial effect of graphene oxide (GO) nano-particles against Pseudomonas putida biofilm of variable age.

Graphene oxide (GO) has been reported to possess antibacterial activity; therefore, its accumulation in the environment could affect microbial communities such as biofilms. The susceptibility of biofilms to antimicrobials is known to depend on the stage of biofilm maturity. The aim of this study was to investigate the effect of GO nano-particles on Pseudomonas putida KT2440 biofilm of variable age. FT-IR, UV-vis, and Raman spectroscopy confirmed the oxidation of graphene while XPS confirmed the high purity of the synthesised GO over 6 months. Biofilms varying in maturity (24, 48, and 72 h) were formed using a CDC reactor and were treated with GO (85 µg/mL or 8.5 µg/mL). The viability of P. putida was monitored by culture on media and the bacterial membrane integrity was assessed using flow cytometry. P. putida cells were observed using confocal microscopy and SEM. The results showed that GO significantly reduced the viability of 48-h biofilm and detached biofilm cells associated with membrane damage while the viability was not affected in 24- and 72-h biofilms and detached biofilm cells. The results showed that susceptibility of P. putida biofilm to GO varied according to age which may be due to changes in the physiological state of cells during maturation. Graphical abstract.

Activity of disinfectants against foodborne pathogens in suspension and adhered to stainless steel surfaces

The purpose of this study was to investigate and compare the efficacy of various disinfectants on planktonic cells and biofilm cells of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Numbers of viable biofilm cells decreased after treatment with all tested disinfectants (iodine, biguanide, quaternary ammonium compounds, peracetic acid and sodium hypochlorite). Sodium hypochlorite was the most effective disinfectant against biofilm cells, while biguanide was the least effective. Scanning electron microscopy observations revealed that cells adhered on stainless steel surface after treatment with the disinfectants. No viable planktonic cells were observed after treatment with the same disinfectants. Based on our findings, we concluded that biofilm cells might be more resistant to disinfectants than plancktonic cells.